Genetic polymorphisms of genes involved in oxidative stress and inflammatory management in oncopediatric patients with chemo-induced oral mucositis

Abstract Oral mucositis (OM) is a painful inflammatory oral condition that affects children who undergo chemotherapy. Oxidative stress is a known OM mediator and pro-inflammatory cytokines contribute to the amplification of the immune response. Objective: To investigate the possible associations of rs4880 (superoxide dismutase 2, SOD2 47 C/T), rs7943316 (catalase, CAT −21 A/T), rs1800629 (tumor necrosis factor α, TNF- α −308 G/A), and rs1800795 (interleukin 6, IL-6 −174 G/C) polymorphisms with chemo-induced OM occurrence and severity in oncopediatric patients. Methodology: We conducted a single-center, observational cross-sectional study with sample collection of oral epithelial cells from 95 children and adolescents with hematological cancers who underwent chemotherapy, followed by genomic DNA extraction. Single-nucleotide polymorphisms (SNPs) were assessed with PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). Demographic data and information concerning OM occurrence were obtained from dental charts of the multidisciplinary oral care team. Information on OM severity was obtained from appropriately-filled Oral Assessment Guide records. Descriptive and inferential statistics were conducted with Student's T test, chi-squared test, and Fisher's exact test, with p≤0.05. Results: The mean age was 10 years-old and most patients were male individuals (57.89%). Female sex was considered a protective factor for OM occurrence (OR=4.83; CI=[1.14; 16.57]). The AA genotype for CAT was the most frequent amongst individuals with severe OM (p=0.04). The GA genotype for TNF- α was the most frequent amongst individuals without severe OM (p=0.03). For SOD2 and IL-6 , the most frequent genotypes were CT and GG respectively for all groups (p>0.05). Conclusion: The AA genotype for CAT −21 A/T was a tendency among the group with severe OM. Data on TNF- α −308 G/A were inconclusive. No associations were detected for SOD2 47 C/T and IL-6 −174 G/C polymorphisms in oncopediatric patients with chemo-induced oral mucositis.

pro-inflammatory cytokines tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6), among others. 5 Superoxide dismutase, alongside catalase enzymes, represents the first line of defense against oxidative damage in living cells 6 and recombinant SOD2 proteins have been demonstrated to attenuate chemo-induced intestinal mucositis in mice. 7 Regarding the cytokines TNF-α and IL-6, both are examples of NF-kBupregulated targets directly involved in OM progression since they are responsible for amplifying the mucosal inflammatory response. 8,9 Their concentration in saliva increases within 4 to 12 days after chemotherapy in adults; these levels were associated with the occurrence of oral lesions. 10 Moreover, elevated levels of plasma TNF-α at the time of diagnosis have been associated with higher risks of developing OM during chemotherapy in acute lymphoblastic leukemia (ALL) pediatric patients. 11 OM prevalence, severity, and form of manifestation varies in the pediatric cancer population, 12,13 making it an interesting target for genetic analysis. Single-nucleotide polymorphisms (SNPs) in genes involving drug metabolism (ABCC2, rs717620; MTHFR, rs1801133; ABCG2 rs2231137) and cell signaling pathways (miR-1206, rs2114358) have been associated with the incidence of chemo-induced OM in pediatric blood cancer patients. [14][15][16][17] However, there are no reports on the relation between SNPs in genes involving antioxidant enzymes and chemo-induced OM in any population. Regarding polymorphisms in cytokine genes in the same context, reports are limited to studies involving adults. 18 Rs4880 (SOD2 47 C/T) is a commonly studied SNP that has been associated with diminished levels of SOD2 19 ; which has been previously studied in the context of periodontal disease, being associated with susceptibility to treatment response. 20

Eligibility criteria and OM diagnosis
Children and adolescents included in this study were those with primary diagnosis of leukemia or lymphoma who were being, or had been previously, submitted exclusively to chemotherapeutical treatment and who had been assessed for oral alterations by the multidisciplinary oral care team during treatment.
Exclusion criteria comprised patients without registries of oral examination by a calibrated professional of the oral care team during treatment, patients that were in no condition to perform sample collection (e.g.: in isolation, intubated, or severely debilitated) or whose caregiver did not consent to it, and those who were treated with a combination of chemo and radiotherapy.

Statistical analysis
For each polymorphism, the Hardy-Weinberg equilibrium (HWE) was obtained using the chi-squared (χ 2 ) goodness-of-fit test. Demographic data were analyzed using Student's T test for continuous variables (age) and χ 2 or Fisher's exact test for categorical ones (sex). For association analysis between genotype or allele frequency and OM occurrence/severity, χ 2 and Fisher's exact tests were performed when appropriate.
The odds ratio and corresponding confidence interval were calculated, when possible, for every association.
Data analysis was conducted using the BioEstat 5.3 software (Instituto Mamirauá, Tefé-AM, Brazil) with a significance level of 5% and considering a confidence interval of 95%. Any p-values <0.05 were considered significant.

Characteristics of the studied population
The sample was comprised of 95 patients ranging from 03 to 19 years of age (mean age = 10.36 ± 4.84 years), in which most were male individuals (57.89%).
Six types of cancer were present in our sample and the most frequent type was ALL (74.73%) ( Table 1).
After sample collection, the study power was estimated at 6.6% for rs4880, 14

Polymorphism frequency and OM occurrence
Out of the 4 polymorphisms investigated in this research, only one, the rs4880 (SOD2 47 C/T), was not in accordance with the HWE (p<0.05). Table 2 shows the genotypic and allele frequencies of all 4 polymorphisms for every association analysis.
Regarding the analysis between the polymorphism frequency and OM occurrence, groups I and II were considered. The comparison between groups did not show any significant differences (p>0.05). For SOD2, a higher frequency for the T allele and CT genotype was observed for both groups. For CAT, the AT genotype was the most frequent in the entire population and, although no significant differences were observed, the T allele was most frequent in the control group while the A allele prevailed in the OM group. For both TNF-α and IL-6, the G allele and the GG genotype were the most prevalent in the entire population. Figure 1 shows the band patterns after electrophoresis.

Polymorphism frequency and OM severity
Regarding the analysis between the polymorphism frequency and OM severity, groups I, III, and IV were considered. Data on OM severity was available for 43 children, out of which 46.51% presented the severe form of the disease (n=20) ( Table 3).
For CAT, the comparison between genotypic frequencies of groups I, III, and IV did not show significant differences. However, when partitioning the χ 2 contingency and IL-6, the most prevalent genotypes were CT and GG respectively (p>0.05) and no differences were detected. in Figure 1 shows the band patterns after electrophoresis.

Discussion
OM is a side effect of cancer treatment, which has been associated with increased use of hospital resources and higher hospitalization costs. 2   Our study focused on exploring different genetic polymorphisms than those commonly analyzed in the context of chemo-induced OM, which are usually related to drug metabolism. [14][15][16] To our knowledge, this is the first study investigating rs4880, rs7943316, Regarding demographic data, our sample was considerably young, with a mean age of 10 years-old.  In regard to the genes involved in oxidative stress management, it was observed that the rs4880 (SOD2) frequencies were not in accordance with the HWE, similarly to other studies involving Brazilian populations. [36][37][38] The CT genotype and T allele for this SNP were the most frequent in our population.
The allelic frequencies of group II are comparable to those of the hypercholesterolemia group (C=37.5%; T=62.5%) from a study by Duarte, et al. 39 (2010) in which this SNP was associated to the condition and to lower MnSOD activity. Although no associations between this SNP and OM occurrence/severity were detected in our study, a reduced enzymatic levels in our population would be expected, leading to an Group I = individuals who did not present OM during the induction phase of treatment. Group III =individuals from group II with a mild case of OM or no OM in the first 60 days of treatment. Group IV = individuals from group II with severe OM in the first 60 days of treatment. For the OM severity analysis, only patients from group II with a thoroughly-filled OAG severity grading scale recorded in their dental charts were included. *χ2 test; ≠Fisher's Exact Test.  shown that the dominant model AA versus AT + TT is associated with risk of preeclampsia. 41 The rs7943316 consists of a A→T substitution in position 21 of CAT promoter region and is associated with diminished expression levels. 21 It should be considered that the promoter region of CAT may be epigenetically modulated by CpG island hypermethylation when submitted to prolonged exposure to ROS. 42 Therefore, higher levels of oxidative stress could still be observed even in the absence of the rare allele for rs7943316 due to epigenetic downregulation of CAT expression.
For the pro-inflammatory cytokines' genes, the GA genotype for rs1800629 (TNF-α) was more frequent in patients without OM (group I; p=0.03, χ 2 test) as well as in patients with a mild case of OM or no OM in the first 60 days of treatment (group III; p=0.0307, χ 2 test) when compared with those with severe OM (group IV). The A allele was also slightly more frequent in group I (13.33%) when compared to group II (5%), but no significant differences were found. The observed p-values suggest that bearing the GA genotype is associated with not developing severe cases of OM. size is expected, resulting in low study powers.
Moreover, for patients treated with chemotherapy, length of treatment has been cited as an important factor when considering duration and severity of OM, among other aspects. 46 Therefore, we limited our analysis to those patients who had thorough records of OM assessment with a complete OAG evaluation in the first 60 days of treatment in order to reduce bias due to drug accumulation. This resulted in small sample sizes that may not have been able to reflect possible associations. Other limitations of this study include relying on the correct filling of medical and dental charts by the multidisciplinary team as well as the lack of information concerning some of the patients. Another important point to discuss is that the Brazilian population is highly mixed with a unique

Conflict of interest
All authors declare no conflict of interest.